Method for obtaining pure monosialoganglioside gm1 for medical use

ABSTRACT

A process for preparing pure monosialoganglioside GM1 in the form of its sodium salt. There is provided a process for the isolation and purification of monosialoganglioside GM1 comprising (a) separation of GM1 from a lipidic mixture containing the monosialoganglioside GM1 as the main ganglioside component by ion exchange column-chromatography using an eluent comprising potassium or caesium ions, (b) recovery of the solute from the eluted solution, (c) diafiltration of an aqueous solution of the recovered solute, and (d) second diafiltration after the addition of 1 M NaCl, and recovering GM1. The purity level of GM1 obtained is higher than 99.0%.

The present invention relates to the monosiaganglioside GM1, moreparticularly to processes for the obtaining and purifying themonosialoganglioside GM1.

BACKGROUND

Gangliosides are a class of glycosphingolipids, having one or moresialic acid residues, and are found abundantly in the cerebral andnervous tissue of humans and animals. The monosialoganglioside GM1 isknown for a number of pharmaceutical applications, particularly in therepair and treatment of disorders of the central and peripheral nervoussystems.

Gangliosides are conveniently extracted from bovine or porcine cerebraltissue according to U.S. Pat. No. 4,849,413. In order to be suitable forpharmaceutical applications, the monosialoganglioside GM1 must then beisolated and purified.

It is known to treat the extracted lipid mixture by chemical orenzymatic methods to transform other ganglioside components to themonosialoganglioside GM1, in order to increase the yield ofmonosialoganglioside GM1. Such methods include acid hydrolysis with aweak acid, such as described in CN 1353112, or enzymatic hydrolysisusing a sialidase, for instance as described in U.S. Pat. No. 5,296,360.

Further purification of such GM1 enhanced lipidic mixture by exclusionchromatography using a chloroform:methanol:water 60:30:4.5 eluent isdescribed in EP 0 150 712. EP 0 489 352 describes purification of a GM1enhanced lipidic mixture by ultra-filtration of a solution of thelipidic mixture with alpha-cyclo-dextrin, followed by extraction of GM1by solvent extraction with ethanol. It is reported that GM1 may beobtained with a purity of 95%.

Such processes have previously been shown to have drawbacks with respectto the purity and yield of GM1, and with respect to cost, efficiency andeffectiveness when applied on an industrial scale.

For pharmaceutical applications it is required to produce theganglioside GM1 at high purity. Accordingly, there remains an ongoingneed for processes for obtaining the ganglioside GM1 at high-purity.

The inventors of present application have surprisingly found that theganglioside GM1 may be effectively separated from other gangliosides bya process based on ion-exchange chromatography.

According to the present invention, it has been found that theganglioside GM1 may be prepared at high purity by a process wherein GM1is separated from a lipidic mixture containing the monosialogangliosideGM1 as the main ganglioside component by ion exchangecolumn-chromatography using an eluent comprising potassium or caesiumions.

According to the present invention there is provided a process for theisolation and purification of the ganglioside GM1 according to claim 1.

According to a preferred embodiment of the present invention there isprovided a process comprising the general steps of:

-   (a) separating GM1 from a lipidic mixture containing the    monosialoganglioside GM1 as the main ganglioside component by ion    exchange column-chromatography using an eluent comprising potassium    or caesium ions,-   (b) recovery of the solute from the eluted solution,-   (c) diafiltration of an aqueous solution of the recovered solute,-   (d) addition of a sodium salt and diafiltration of the resultant    solution, and-   (e) recovery of GM1.

Advantageously the process of the present invention allows for thepreparation of the monoganglioside GM1 at high purity. According to thepresent invention the monosialoganglioside GM1 may be prepared with apurity of higher than 98%, even higher than 99.0% and even 99.9%.

Further, the process of the present invention advantageously uses simplesteps, is cost-efficient and is suitable for application on anindustrial scale.

Other objects and advantages of the present invention will be apparentfrom the claims and from the following detailed description andexamples.

DETAILED DESCRIPTION

The present invention provides a process for the purification of themonoganglioside GM1, wherein GM1 is separated from a lipidic mixturecontaining the monosialoganglioside GM1 as the main gangliosidecomponent by ion exchange column-chromatography using an eluentcomprising potassium or caesium ions.

In a preferred embodiment, there is provided a process for preparingmonosialoganglioside GM1 at high purity comprising the steps of;

-   (a) separation of GM1 from a lipidic mixture containing the    monosialoganglioside GM1 as the main ganglioside component by ion    exchange column-chromatography, using an eluent comprising potassium    or caesium ions.-   (b) recovery of the solute from the eluted solution of step (a),-   (c) diafiltration of an aqueous solution of the recovered solute of    step (b), in order to eliminate residual potassium or caesium salts,-   (d) addition of sodium ions, preferably in the form of a suitable    sodium salt, in order to displace the potassium or caesium ions    bound to GM1, and diafiltration of the aqueous solution, in order to    eliminate residual sodium salt, and-   (e) recovery of GM1, in the form of its sodium salt.

The lipidic mixture may be prepared from crude lipid extract of bovine,ovine, equine or porcine cerebral tissue.

Advantageously, the lipidic mixture containing the monoganglioside GM1as the prevailing ganglioside component may be prepared from a lipidextract containing at least 30%, preferably at least 50% and morepreferably at least 70% of a mixture of gangliosides. The remainder ofthe lipid extract may generally be composed of sulfatides, cerebrosides,fatty acids and proteins.

The lipid extract may advantageously be first subjected to adiafiltration through a membrane having pore size of 10000 to 100000Daltons, preferably about 50000 Daltons, in order to desalt thesolution. For the diafiltration any conventional dialysis membrane maybe used. Advantageously filter cassettes, e.g. of the SARTOCON®(Sartorius) polysulfone cassettes type may be used, for example with acut-off of about 50000 Daltons.

Preferably the lipid extract is subject to treatment by hydrolysis totransform other major ganglioside components, such as GT1b, GD1a, andGD1b, into GM1 in order to increase the GM1 content.

The hydrolysis may be carried out using conventional methods.Advantageously the hydrolysis may be carried out using either of twogeneral methods for hydrolysis of gangliosides known in the literature,namely acid hydrolysis or enzymatic hydrolysis.

Acid hydrolysis may be carried out, for example, using dilute mineralacid, such as dilute hydrochloric acid, sulphuric acid and nitric acid.The acid hydrolysis may be effected by adjusting the pH of an aqueoussolution of the lipid extract to a pH between 3.5 and 5, preferablyaround pH 4.0, and heating the solution to a temperature preferablybetween 90° C. and 100° C. for the time necessary to complete theconversion of the other major ganglioside components to GM1. The timeneeded to hydrolyze the major gangliosides to GM1 depends on the pH andtemperature chosen. In general, the higher the pH the longer the timerequired to complete the hydrolysis, and the higher the reactiontemperature the shorter the time required to complete the hydrolysis.The hydrolysis reaction may generally be carried out over 2 to 5 hours.For example, where the hydrolysis is carried out at pH 4.0 and 95° C.,the time required to complete the hydrolysis reaction is about 3 hours.

Enzymatic hydrolysis may be carried out using any suitable sialidase.Preferably Arthrobacter ureafaciens sialidase strain S or Vibriocholerae sialidase may be used. Advantageously, Arthrobacter ureafacienssialidase strain S and Vibrio cholerae sialidase are active on GT1a,GD1a, GD1b but not on GM1. Enzymatic hydrolysis may be carried out, forexample, by adjusting the pH of an aqueous solution of the lipid extractto a pH at which the enzyme used has its optimum activity, for instancebetween pH 4 and pH 6, for example about pH 5, by adding a suitablebuffer, such as acetate buffer, adding Ca2+ ions in the case that thesialidase is a Vibrio cholerae sialidase, and heating the solution at atemperature at which the enzyme used has its optimum activity, forexample around 37° C., for the time need to complete the transformation.The hydrolysis may generally be carried out over 12-24 hours, dependanton the enzyme units added.

Acid hydrolysis is less preferred as it is a non-specific hydrolysisprocess and generally provides a lower yield in GM1 due to conversion toother gangliosides. Further the acid hydrolysis process leads to theformation of asialo-GM1 impurity.

The enzymatic hydrolysis methods are preferred as they generally providea higher yield in GM1 due to the high specificity of the chemicaltransformation. For the enzymatic hydrolysis, Arthrobacter ureafacienssialidase is preferred as it does not require the addition of Ca2+ ionsfor its activity. Further, due to its molecular weight of 52,000 Daltons(compared to 82,000 Daltons for Vibrio cholerae sialidase), it mayadvantageously be washed out by diafiltration.

In order to recover the thus produced lipidic mixture having an enhancedcontent of the monosialoganglioside GM1 from the reaction solution, thereaction solution may advantageously be diafiltered, e.g. through amembrane having pore size of 10000 to 100000 Daltons, preferably about50000 Daltons. For the diafiltration any conventional dialysis membranemay be used. Advantageously filter cassettes, such as of the SARTOCON®(Sartorius) polysulfone cassettes type, may be used, preferably with acut-off of 50000 Daltons. The permeate may then be dried to obtain apowder of the lipidic mixture containing the monosialoganglioside GM1 asthe main ganglioside component. Drying may be carried out byconventional methods. Advantageously the drying may be carried out byspray drying or vacuum drying.

The lipidic mixture may generally have a concentration of GM1 of 10 to200 g/lt and preferably of at least 100 g/lt.

According to the process of the present invention, the ganglioside GM1is separated from other gangliosides in the lipidic mixture usingion-exchange chromatography.

It has surprisingly been found that where an eluent containing caesiumor potassium ions is used it is possible to successfully separate outGM1 from other monosialogangliosides.

Conventionally used ion exchange techniques have generally been foundnot to allow effective separation of GM1 from othermonosialogangliosides. Of particular note is the monosialogangliosideFucosyl-GM1, which is present as a major ganglioside impurity in theporcine lipidic mixture produced by the known hydrolysis treatments.

The two molecules GM1 and Fucosyl-GM1 have very similar physicalproperties. Both have a single negative charge, provided by the carboxylgroup of the sialic acid, and have similar molecular weights; 1558 and1704 respectively. Accordingly, when loaded onto the pre-equilibratedion-exchanger column, the binding strength of the two gangliosides withthe resin is the same.

It has been observed that when sodium acetate is added to the eluent inorder to increase the ionic strength of the eluent, and provide theconditions for displacement of the gangliosides, both of GM1 andFucosyl-GM1 are eluted at the same time with the same ionic strength. Noseparation of the two monosialogangliosides can be achieved.

Whereas present inventors have surprisingly found that where cesium orpotassium ions are used, for instance by the addition of methanolicpotassium or cesium acetate, GM1 and Fucosyl-GM1 are eluted separately,with Fucosyl-GM1 being eluted first. The separation is complete, andeach of the gangliosides GM1 and Fucosyl-GM1 can be isolated.

Whilst not wishing to be bound by any theory, it is considered by theinventors of the present invention, that the observed separation may beattributed to the fact that, contrary to the conventional ion-exchangetheory, the solutes GM1 and Fucosyl-GM1 are not only released from thecolumn in order of their strength in binding with the resin, but also inorder of their affinity for the counter-ion to which they must bind inorder to be detached from the gel. Accordingly, following this theory,if one of the two solutes has a different affinity for the counter-ion,then the solutes will be released with two different ionic strengths,and purification can occur.

It has surprisingly been found by the present inventors that themonosialogangliosides GM1 and Fuc-GM1 have the same affinity for sodiumbut do not have not the same affinity for potassium or caesium, despitethe fact that all three metals belong to the same group. It has beenfound by the present inventors that Fuc-GM1 has a higher affinity forpotassium and caesium than GM1, and is eluted first.

The ion-exchange chromatography method of the present inventionadvantageously enables effective separation of GM1 from Fucosyl-GM1.Further, the method of the present invention advantageously also allowsthe separation of GM1 from corresponding fatty acids. The fatty acidshaving the same charge as the ganglioside, but a higher affinity forcaesium or potassium ions.

For the ion exchange chromatography, any suitable resin may be used.Advantageously a resin having a quaternary amino group may be used, forexample, FRACTOGEL® EMD TMAE (S) or Sepharose resins e.g. Q-Sepharose HPresins.

In a first stage, the resin is equilibrated with a suitable solvent.Suitable solvents include ethanol, methanol or a mixture of methanol andchloroform. Preferably the solvent is methanol, because it is a solventin which gangliosides and potassium and caesium salts are soluble.

The column may then be loaded with a solution of the lipidic mixture ina suitable elution solvent. The elution solvent should contain the samesolvent components as the solvent used for equilibration of the resin.Preferably the solvent chosen is methanol. Preliminary elution with theelution solvent enables elution of unbound substances, e.g.cerebrosides.

Potassium or cesium ions are then added to the elution solvent. Thepotassium or cesium ions are preferably provided in the form of amethanolic solution of potassium or cesium acetate, formate, proprionateor as a salt of other organic acid. A methanolic solution of sodium orpotassium acetate is preferred. Advantageously potassium or cesiumacetate may be present in the eluent in an amount sufficient to impair aconductivity of 1100-1400 μS/cm, preferably a conductivity of 1200-1300μS/cm on the eluent. This sodium or potassium salt containing eluentsolution may be passed through the column isocratically at any suitableflow rate, for instance at a flow rate between 100 ml/h to 140 ml/h.

Where the separation is carried out using the ion exchangechromatography process according to the present invention, fatty acidsand Fuc-GM1 are eluted before GM1, whilst sulfatides remain bound to thecolumn may be eluted during column washing.

The GM1 containing eluate is collected and, advantageously, the elutionsolvent eluted from the column may be recovered by distillation.

The GM1 containing solute may be recovered by drying the eluate solutionto produce a powder containing the GM1. Drying may be carried out usingconventional methods, for instance spray-drying or vacuum drying.

The GM1 containing solute may then be purified by diafiltration of anaqueous solution thereof through a membrane having pore size of 10000 to100000 Daltons, preferably about 50000 Daltons, in order to eliminateresidual potassium or caesium salts. Optionally, a mineral acid, such asaqueous sulphuric acid, nitric acid, or preferably hydrochloric acid,may be added to the solution to adjust to a pH between pH 6 to 8,preferably about pH 7 before diafiltration.

Sodium ions, suitably in the form of an aqueous solution of a sodiumsalt, preferably NaCl, may then be added to the solution, in order todisplace the potassium or caesium ions linked to GM1, and obtain GM1 inthe form of the physiological sodium salt. The solution may then besubject to a second diafiltration in order to eliminate residual salt(e.g. NaCl), using a membrane having pore size of 10000 to 100000Daltons, preferably about 50000 Daltons. Advantageously filtercassettes, such as cassettes of the SARTOCON® (Sartorius) polysulfonetype, may be used, preferably with a cut-off of 50000 Daltons.

The solution may then be dried to recover the GM1 in the form of apowder. Drying may be carried out by conventional methods.Advantageously the drying may be carried out by spray drying or vacuumdrying.

GM1 obtained according to the present invention has a degree of purityof 98% or more, generally of about 99.0 to 99.9%. The GM1 obtainedaccording to the process of the present invention contains less than0.1% Fucosyl-GM1 impurity.

The process of the present invention advantageously enables efficientseparation of GM1 from other monosialogangliosides. Particularly, theprocess of the present invention allows the separation of GM1 fromFucosyl-GM1 impurity.

Advantageously the process of the present invention allows thepreparation of the monosialoganglioside GM1 with a good yield and a highlevel of purity. Accordingly, the invention includes the purifiedmonosialoganglioside GM1 in a pharmaceutically acceptable carrier, whichpharmaceutical composition is prepared by techniques known to thoseskilled in the art. In one embodiment, the composition is sterile.

The purified GM1 according to the process of the present invention maybe used in the treatment of human, mammal or animal subjects.Particularly, the purified GM1 according to the present invention isenvisaged for the treatment of humans or mammals, particularly for therepair and treatment of disorders and diseases of the central andperipheral nervous systems, including cerebral stroke, Parkinson'sdisease, spinal cord injury, Alzheimer's disease, Tardive Dyskenisia,Amyotrophic Lateral Schlerosis, peripheral neuropathies and autonomicneuropathy. Preferably, a therapeutically effective amount of apharmaceutical composition comprising the purified monosialogangliosideGM1 in a pharmaceutically acceptable carrier is administered to apatient, i.e., a human, mammal or animal in need of treatment.

The present invention is further illustrated by the following examples.

EXAMPLES Example 1 Preparation of A Lipidic Mixture Containing theMonosialoganglioside GM1 As The Principal Ganglioside

Lipid extract containing a mixture of gangliosides having a purity of70% is dissolved in purified water at a concentration of about 25 g/l.This solution is then diafiltered through SARTOCON® (Sartorius)polysulfone filter cassettes having a cut-off of 50 000 Daltons.

200 l of the solution are then equilibrated to pH 5.5 by addition of 50mM acetate buffer and 4 mM of Calcium chloride. 30000 U of Vibriocholerae sialidase are added and the solution heated to 37° C. for 12 hto complete transformation of other major gangliosides (GT1b, GD1a,GD1b) to GM1. The resulting solution has a GM1 concentration of 14-15g/l.

After the enzymatic hydrolysis, the solution is diafiltered throughfilter cassettes having a cut-off of 50 000 Daltons. 1 M NaCl is thenadded to the retentate and the solution subjected to a seconddiafiltration. After the second diafiltration the retentate isconcentrated to a concentration of GM1 of 100 g/l by letting thepermeate flow without any water replacement. Then, the solution is driedunder vacuum to obtain about 3200 g of a powder having a GM1concentration of 85% measured by HPLC.

Purification of GM1 From Lipidic Mixture Containing theMonosialoganglioside GM1 As the Principal Ganglioside

A methanolic solution at a concentration of 10 g/l is prepared using thepowder obtained in the previous step, and the solution is filteredthrough a 0.22 μm Sartopore cartridge filter (manufactured by SartoriusAG).

For each cycle, 7 litres of the filtered solution are then loaded on anFPLC column containing 20 l of Fractogel® EMD TMAE (S) resinequilibrated in methanol. The column is then eluted with methanol:potassium acetate methanolic solution having a conductivity of 1200-1300μS/cm, at a flow rate of 120 l/h. The cycles repeated until the end ofthe GM1 solution.

The eluate (about 60-70 l) is continuously concentrated by distillation,and is then dried to obtain a powder, and methanol is recovered. Thethus-obtained powder is a mixture of pure GM1 and potassium acetate.

The thus-obtained powder is dissolved in purified water to aconcentration of 25g/l, and equilibrated to pH 7 by the addition of 18%HCl. The solution is diafiltered through filter cassettes having acut-off of 50,000 Daltons. 1M NaCl is then added and the solution isagain diafiltered through filter cassettes having a cut-off of 50,000Daltons. After this second diafiltration the retentate is concentratedup to 100-120 g/l by letting the permeate flow without any waterreplacement.

The concentrated solution is then filtered through a 0.22 μm filter anddried by spray drying to provide about 2700 g of a white to white-beigepowder of GM1, identified by TLC, this GM1 powder having a purity of99.8% measured by HPLC. The resultant GM1 powder has a Fuc-GM1 contentof less than 0.1%, measured by HPLC.

Comparative Example

The process for preparing and purifying GM1 was carried out as inExample 1 above, except that in the column chromatography, the methanol:potassium acetate methanolic solution was replaced by a methanol: sodiumacetate methanolic solution.

3100 g of a powder of GM1 was obtained containing 91% GM1 and 8%Fuc-GM1, measured by HPLC.

From the above examples it can be seen that a much lower purity of GM1is obtained where sodium acetate is used for the elution of GM1. Thismay be attributed to the fact that the sodium counter-ion does not makeany difference between GM1 and Fuc-GM1 in the elution process, comparedwith potassium or caesium counter-ions, which on the contrary completelyseparate both peaks.

All publications, patents, and patent applications identified above areincorporated herein by reference in their entirety.

Although this invention has been described in relation to certainpreferred embodiments thereof, and many details have been set forth forpurposes of illustration, it will be apparent to those skilled in theart that the invention is susceptible to additional embodiments and thatcertain of the details described herein may be varied considerablywithout departing from the basic principles of the invention.

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 13. A composition, comprising; amonosialoganglioside GM1, wherein said monosialoganglioside GM1 l ispurified by separating said monosialoganglioside GM1 from a lipidicmixture containing the monosialoganglioside GM1 as the main gangliosidecomponent, by ion exchange column-chromatography using an eluentcomprising potassium or caesium ions.
 14. The composition according toclaim 13, comprising less than 0.1% Fuc-GM1.
 15. The compositionaccording to claim 13 further including at least one pharmaceuticallyacceptable carrier.
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 18. (canceled)
 19. Thecomposition according to claim 13, wherein said monosialoganglioside GM1is at least 99.0% pure.